In this application, the applicant is seeking support to continue studies of the structure of the Na+/K+-translocating ATPase of animal cell plasma membranes. The NaK-ATPase, a member of the P-type ATPase family of ion-translocating ATPases, catalyzes the active transport of Na+ and K+ and is the receptor for inotropic cardiac glycosides in the heart. Much is known about its enzymatic mechanism, but there are major gaps in understanding its structure, including that of its cardiac glycoside binding site. Antibodies and other proteins that interact with the native, unmodified protein have been used to test models of the transmembrane topology of this transporter. Dr. Sweadner now proposes to obtain new monoclonal antibodies against the sites where the structure is most uncertain, the extracellular surface and the C-terminal third of the a subunit. The antibodies will be characterized by determining whether they bind to the intracellular or extracellular surfaces, by mapping their epitopes, and by measuring any effects on the function of the enzyme. The properties of each antibody will determine which of four principal scientific aims it will be used for. First, mapped antibodies will be used to discriminate between different hypothetical folding models to determine the enzyme's topology. Second, the hypothesis will be tested that the C-terminal third of the a subunit is a folding domain with unique, conformation-dependent structural lability. Thermal denaturation, coupled with the use of antibodies as probes of native and denatured structure, will be used to define the structural domains and build a model of active transport. Third, antibodies against extracellular sites will be used to probe the structure of the cardiac glycoside binding site, to determine whether it is superficial or buried in a pocket. Finally, extracellular antibodies that bind preferentially to the native enzyme will be evaluated for binding to the enzyme in detergent. The variable domains (Fv) will be cloned from the hybridomas and expressed in E. coli with an affinity tag. The modified Fv fragments will be tested for enzyme purification and stabilization, with the long-term goal of co-crystallizing the antibody fragment with the NaK-ATPase. In sum, a combination of hybridoma technology, protein chemistry, and molecular approaches will be used to investigate the structure of this important plasma membrane protein.